Botulinum neurotoxins (BoNTs), synthesized by the Gram-positive, soil-dwelling bacterium Clostridium botulinum, are the most toxic substances known to humankind and are the causative agents of the neuroparalytic disease botulism (Johnson E (2005) in Topley and Wilson's microbiology and microbial infections, ed S. P. Borriello, P. R. Murray, and G. Funke (Hodder Arnold, London, United Kingdom), pp 1035-1088). Seven immunologically distinct serotypes of BoNTs designated A through G have been described (Gimenez D F & Gimenez J A (1995) Int J Food Microbiol 27: 1-9). BoNTs are initially synthesized as a single-chain polypeptide of ˜150 kDa, but posttranslational proteolytic cleavage yields distinct heavy and light chains (HC and LC) of ˜100 kDa and ˜50 kDa linked by a disulfide bond. The HC is further functionally divided into the HCC and HCN sub-domains. The HCC domain is responsible for recognition and binding to specific neuronal cell surface receptors leading to endocytosis, while the HCN domain is responsible for channel formation in the endocytic vesicle membrane and translocation and internalization of the LC across the endosomal membrane (Montecucco et al., (2004) Trends Microbiol 12: 442-446; Fischer A & Montal M (2007) J Biol Chem 282: 29604-29611; Fischer A, et al (2009) Proc Natl Acad Sci USA 106: 1330-1335). During translocation, the disulfide bond is cleaved, and the LC is released into the cell cytosol and refolded to the active enzyme component as a zinc-dependent endopeptidase (Fischer et al., supra; Fischer A & Montal M (2007) Proc Natl Acad Sci USA 104: 10447-10452). The LC then specifically targets and cleaves an intracellular SNARE protein at the pre-synaptic vesicles, which leads to inhibition of neurotransmitter release. Each BoNT serotype has a distinct cleavage target, with BoNT/A and E cleaving SNAP-25 at distinct sites, BoNT/B, D, F, and G cleaving VAMP/synaptobrevin at different sites, and BoNT/C cleaving both SNAP-25 and syntaxin (reviewed in Montecucco C & Schiavo G (1994) Mol Microbiol 13: 1-8).
Naturally occurring botulism is a rare but serious disease, with ˜110 cases occurring per year in the United States and a lethality rate of ˜5-10% (Johnson E A & Montecucco C (2008) in Handbook of Clinical Neurology, ed Andrew G. Engel (Elsevier, pp 333-368). Due to their extreme potency (estimated human lethal dose of 1 ng/kg of body weight for BoNT/A (Bossi P, et al (2006) Cell Mol Life Sci 63: 2196-2212), the severity of the disease botulism and the high cost involved in treating cases, especially at a large scale, BoNTs have been classified as a category A Select Agent and present a serious threat as a bioterrorism weapon (Arnon S S, et al (2001) JAMA 285: 1059-1070).
BoNT/A and to a much lesser extent BoNT/B are also being used as unique and important pharmaceuticals to treat a variety of neuromuscular disorders and in cosmetics. Conditions for which the Food and Drug Administration approved the use of BoNTs include cosmetic treatments and to temporarily relieve a variety of muscle spasticity disorders, hyperhydrosis and migraines (Chaddock J A & Acharya K R (2011) FEBS J 278: 899-904). Cosmetic and clinical applications of BoNTs are increasing, and new formulations of BoNTs for pharmaceutical purposes are being developed necessitating clinical trials, accurate potency determination, and neutralizing antibody screening. For example, BoNTs are pharmaceutically administered for the treatment of pain disorders, voluntary muscle strength, focal dystonia, including cervical, cranial dystonia, and benign essential blepharospasm, hemifacial spasm, and focal spasticity, gastrointestinal disorders, hyperhidrosis, and cosmetic wrinkle correction, Blepharospasm, oromandibular dystonia, jaw opening type, jaw closing type, bruxism, Meige syndrome, lingual dystonia, apraxia of eyelid, opening cervical dystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, spasmodic dysphonia/adductor type, spasmodic dysphonia/abductor type, spasmodic dyspnea, limb dystonia, arm dystonia, task specific dystonia, writer's cramp, musician's cramps, golfer's cramp, leg dystonia, thigh adduction, thigh abduction knee flexion, knee extension, ankle flexion, ankle extension, equinovarus, deformity foot dystonia, striatal toe, toe flexion, toe extension, axial dystonia, pisa syndrome, belly dancer dystonia, segmental dystonia, hemidystonia, generalised dystonia, dystonia in lubag, dystonia in corticobasal degeneration, dystonia in lubag, tardive dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in Hallervorden-Spatz disease, dopa-induced dyskinesias/dopa-induced dystonia, tardive dyskinesias/tardive dystonia, paroxysmal dyskinesias/dystonias, kinesiogenic non-kinesiogenic action-induced palatal myoclonus, myoclonus myokymia, rigidity, benign muscle cramps, hereditary chin trembling, paradoxic jaw muscle activity, hemimasticatory spasms, hypertrophic branchial myopathy, maseteric hypertrophy, tibialis anterior hypertrophy, nystagmus, oscillopsia supranuclear gaze palsy, epilepsia, partialis continua, planning of spasmodic torticollis operation, abductor vocal cord paralysis, recalcitant mutational dysphonia, upper oesophageal sphincter dysfunction, vocal fold granuloma, stuttering Gilles de la Tourette syndrome, middle ear myoclonus, protective larynx closure, postlaryngectomy, speech failure, protective ptosis, entropion sphincter Odii dysfunction, pseudoachalasia, nonachalsia, oesophageal motor disorders, vaginismus, postoperative immobilisation tremor, bladder dysfunction, detrusor sphincter dyssynergia, bladder sphincter spasm, hemifacial spasm, reinnervation dyskinesias, cosmetic use craw's feet, frowning facial asymmetries, mentalis dimples, stiff person syndrome, tetanus prostate hyperplasia, adipositas, treatment infantile cerebral palsy strabismus, mixed paralytic concomitant, after retinal detachment surgery, after cataract surgery, in aphakia myositic strabismus, myopathic strabismus, dissociated vertical deviation, as an adjunct to strabismus surgery, esotropia, exotropia, achalasia, anal fissures, exocrine gland hyperactivity, Frey syndrome, Crocodile Tears syndrome, hyperhidrosis, axillar palmar plantar rhinorrhea, relative hypersalivation in stroke, in Parkinsosn's, in amyotrophic lateral sclerosis, spastic conditions, in encephalitis and myelitis autoimmune processes, multiple sclerosis, transverse myelitis, Devic syndrome, viral infections, bacterial infections, parasitic infections, fungal infections, in hereditary spastic paraparesis postapoplectic syndrome hemispheric infarction, brainstem infarction, myelon infarction, in central nervous system trauma, hemispheric lesions, brainstem lesions, myelon lesion, in central nervous system hemorrhage, intracerebral hemorrhage, subarachnoidal hemorrhage, subdural hemorrhage, intraspinal hemorrhage, in neoplasias, hemispheric tumors, brainstem tumors, and myelon tumor. Thus, the quantitative and reliable detection of BoNT activity in the environment, in foods, in pharmaceutical preparations, for antibody detection, and in research applications is crucial in both prevention of botulism, for counter-terrorism, as well as new drug development and patient safety and quality control and assurance testing of products.
Many BoNT detection methods have been published, and they can be divided into four general categories (reviewed in Cai et al., (2007) Crit Rev Microbiol 33: 109-125): 1. in vitro assays that immunologically detect the presence of holotoxin but cannot distinguish between active or inactive states (ELISA); 2. endopeptidase assays that detect the enzymatic activity of the toxin LC but do not distinguish between biologically active holotoxin and the LC only; 3. in vivo assays (mouse bioassay); and lastly 4. in vivo simulation assays such as the hemidiaphragm assay, local injection assays, and cell-based assays using primary or immortalized cells. In order to detect fully active BoNTs, a detection assay should measure all steps of the intoxication process (e.g., HC binding to the cell surface receptors, endocytosis, vesicle channel formation, cleavage of the disulfide bond, transduction of the LC into the cell cytosol, and finally proteolytic cleavage of SNARE proteins). Only the mouse bioassay and the in vivo simulation assays measure all of these steps. The mouse bioassay involves injecting mice either intravenously or intraperitoneally with different dilutions of BoNT, and then observing the mice for symptoms of botulism poisoning (limb paralysis, labored breathing, ruffled fur, etc.) (Hatheway C L (1988) in Laboratory diagnosis of infectious diseases: principles and practice. eds Balows A, Hausler W H, Ohashi M & Turano M A (Springer-Verlag, New York), pp 111-133; Schantz EJaK, D. A. (1978) Journal of the Association of Official Analytical Chemists 61: 96-99) and ultimately death. Although the MBA is quantitative and can monitor all the steps of intoxication, it has a large error rate, is not standardized between or within labs, requires a large number of animals, and the corresponding facilities and trained staff. The hemidiaphragm and local injection assays reduce the suffering of animals and some are sufficiently sensitive, but still require large numbers of animals and skilled staff.
These clearly identified shortcomings of these assays have incited a recommendation from regulatory agencies including the FDA and USDA to develop a cell-based model that would provide a specific, sensitive, and quantitative alternative to the MBA (National Institute of Environmental Health Sciences, 2008). Various continuous cell lines, including neuro-2a and PC-12, have been used for toxicity testing, but are not sensitive enough to compete with the MBA. Primary neurons derived from rat, mouse, or chicken, and neurons derived from mouse embryonic stem cells are significantly more sensitive (Hall Y H, et al (2004) J Immunol Methods 288: 55-60; Keller J E, Cai F & Neale E A (2004) Biochemistry 43: 526-532; Lalli G, et al (1999) J Cell Sci 112 (Pt 16): 2715-2724; Neale et al., (1999) J Cell Biol 147: 1249-1260; Stahl A M, et al (2007) J Biomol Screen 12: 370-377). The most sensitive cell type for toxicity testing and antibody detection described is the primary rat spinal cord cells (RSC) assay (Pellett et al., (2007) FEBS Lett 581: 4803-4808), which is more sensitive than the MBA, reproducible, and correlates well with the mouse bioassay (Pellett et al., (2010) J Pharmacol Toxicol Methods). Additionally, neurons derived from embryonic stem cells have also been shown to be highly sensitive (McNutt et al., (2011) Biochem Biophys Res Commun 405: 85-90; Pellett S, et al (2011) Biochem Biophys Res Commun 404: 388-392; Kiris E, et al (2011) Stem Cell Res). However, the RSC assay still requires the use of some animals and skilled staff for cell preparation, and is not easily adaptable to testing standardization due to the need to continuously prepare new batches of cells.